Anti-tumor properties of Dickkopf 3b

ABSTRACT

The invention relates to novel therapeutic approaches to cancer treatment that exploits tumor suppressor functions of DKK3b by site-specific delivery of DKK3b. Novel therapeutics and methods for treating tumors and cancers utilizing DKK3b tumor suppressor functions are disclosed.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/157,334, filed May 17, 2016, now U.S. Pat. No.10,407,478, which is a continuation application of U.S. patentapplication Ser. No. 14/511,667, filed Oct. 10, 2014, now U.S. Pat. No.9,856,301, which claims the benefit of priority, under 35 USC 120, fromthe US designation of international Application No. PCT/US13/31118,filed on Mar. 14, 2013, which claims benefit of priority from U.S.Provisional Application Ser. No. 61/615,514, filed on Mar. 26, 2012. Theentire contents of each application is incorporated herein by referencein its entirety for all purposes.

GOVERNMENT RIGHTS

This invention was made with Government support under grant nos.DK038772 and DK060051 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to tumor suppressor properties andfunctions of DKK3b and anti-tumor applications and uses thereof. Moreparticularly, the invention relates to novel therapeutics and methodsfor treating tumors and cancers utilizing DKK3b tumor suppressorfunctions, for example, via site-specific delivery of DKK3b or a relatedagent.

BACKGROUND OF THE INVENTION

Suppressed Dickkopf-3 (DKK3) expression is a hallmark of many humancancers and expression levels are inversely related to tumor virulence(e.g., in prostate cancer and ovarian cancer). Using prostate cancer asan example, over-expression of DKK3 halts proliferation of prostatecancer cells, but the beneficial consequences of DKK3 over-expression inboth in vivo and ex vivo models of prostate cancer are likely anartifact of the inadvertent initiation of an ER stress response in cellsattempting to process an over-expressed, exogenous, secretory geneproduct. (Abarzua, et al. 2005 Cancer Res 65(21): 9617-22; Abarzua, etal. 2008 Biochem Biophys Res Commun 375(4): 614-8; Abarzua, et al. 2007Int J Mol Med 20(1): 37-43.)

The tumor suppressor activity of DKK3 was also reported to be due to itsability to block the translocation of β-catenin to the nucleus byforming an inactive complex composed of a cytoplasmic ˜30 kDa DKK3 geneproduct and βTrCP. (Lee, et al. 2009 Int J Cancer 124(2): 28797.) Sinceit is unlikely that chronic ER stress is the mechanism by whichendogenous DKK3 gene products influence normal cell proliferation, theidentification of a non-secreted, intracellular version of DKK3 and thediscovery of two intracellular events (JNK activation and β-catenininactivation) that facilitate growth arrest offer an opportunity todefine the molecular events mediating the DKK3 tumor suppressor functionin the prostate.

Early studies by Dr. Leonard and colleagues discovered that the DKK3gene locus encodes a second transcript that produces an intracellularmembrane associated 29 kDa protein (formally D2p29, now renamed, DKK3b).(Leonard, et al. 2000 J Biol Chem 275(33): 25194-201; Farwell, et al.1996 J Biol Chem 271(27): 16369-74; Safran, et al. 1996 J Biol Chem271(27): 16363-8; Farwell, et al. 1993 J Biol Chem 268(7): 5055-62.)

Subsequent studies revealed that this DKK3b gene product originated froma second transcriptional start site located in intron 2. Dkk3b wasoriginally identified in astrocytes as a highly trafficked membraneprotein that binds thyroid hormone with high affinity. Analysis of theDKK3 gene locus revealed that DKK3b is encoded by exons 3-8 and that afunctional transcriptional start site—with a TATA box—is located inintron 2 (FIG. 1A). Promoter mapping studies narrowed the promoteractivity to ˜250 bases upstream of exon 3. Deletion of the TATA boxblocked promoter function. ChIP analysis revealed that this promoter wasfunctional in vivo (FIG. 1B).

Importantly, in both the full length DKK3 and truncated DKK3b mRNAs, theonly authentic Kozak start site is located at the Met beginning at exon3, and in vitro translation of the full-length DKK3a mRNA using Kozakcontext dependent conditions yields a 29 kDa protein. (Leonard, et al.2000 J Biol Chem 275(33): 25194-201.) Real time PCR analysis of DKK3locus transcripts revealed that the DKK3a transcript (exons 2-8)accounted for ˜55% of the total DKK3 mRNAs, while the DKK3b transcript(exons 3-8) contributed ˜45% of the total DKK3 mRNAs. In the ΔDKK3 mousethat lacks exons 2, full-length DKK3a transcripts are lost, but theDKK3b mRNA is preserved (FIG. 1C). (Barrantes, et al. 2006 Mol Cell Biol26(6): 2317-26.) Affinity labeling of DKK3b associated with the cellularmembranes of ΔDKK3 mouse brain yielded the anticipated immuno-reactiveDKK3b, while a full-length glycosylated DKK3 was not synthesized (FIG.3D). (Farwell, et al. 1989 J Biol Chem 264(34): 20561-7.) These datademonstrate that the DKK3 locus encodes two functional transcripts; oneencoding a secreted glycoprotein identified as DKK3a, and another anintracellular 29 kDa DKK3b protein.

There remains an ongoing need for establishing novel therapeuticapproaches to cancer treatment utilizing DKK3b tumor suppressorfunction, for example, treatment methodologies and pharmaceuticalcompositions that can arrest the growth of various cancers, such asovarian and prostate cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts exemplary characterization of the mouse Dkk3 gene.

FIG. 2 depicts exemplary effects of DKK3a and DKK3b on PC3 cellproliferation. PC3 cell lines harboring the tet-regulated DKK3a ortet-regulated DKK3b were seeded into 96 well microtiter dishes. At thestart of the experiment, replicate wells (n=5) were fed with growthmedium ±100 ng/ml tet and cell number was determined using the WST-1reagent. Each data point represents the mean of quintuplets; 3 repeattime course experiments are shown.

FIG. 3 depicts exemplary effects of increasing DKK3b on cellproliferation in Du145 cells. DU145 cells carrying the tet-inducibleDKK3b were seeded into 96 well microtiter dishes. At t=0 tet was addedas indicated and cell proliferation was measured using WST-1. Data aremeans, n=5.

FIG. 4 depicts exemplary gene editing scheme for the Dkk3b promoter.

FIG. 5 depicts exemplary, A. Effect of JNK inhibition on DKK3b-inducedcell loss; B. Ploidy analysis in control and ΔJnk MEF; and, C. grossmorphology of prostate tumors in wildtype, ΔJnk, ΔPten and ΔPten/ΔJnkmice. A. DU145 cells expressing DKK3b were treated without or with theJNK inhibitor TAT-JBD for 48 h. Cell proliferation determined as in FIG.2. B. Data from FACS with >30,000 PI-stained cells gated on >4n cells;p=passage number. C. Prostates collected at 20 weeks of life.

FIG. 6 depicts exemplary DKK3b and β-catenin expression at early andlate stages of mammary tumor formation in TBP mouse. Hollow arrow,normal epithelium; solid arrow, early tumor cells; shaded arrow,β-catenin high/DKK3b low; asterisk, DKK3b high/β-catenin low.

FIG. 7 depicts exemplary Dkk3b shows variable expression among mousetumor derived cell lines. Cell lines 1055, 1029, 1113T and K5957 werederived from primary tumors of the TBP knockout mouse. NMuMG areimmortalized normal mammary epithelium.

FIG. 8 depicts exemplary exogenous Dkk3b but not Dkk3a suppressesproliferation of immortalized NMuMG mouse mammary cells (p<0.002).

FIG. 9 depicts exemplary Wnt pathway inhibitor, XAV939, suppressedtumorsphere formation of five independent TBP tumor-derived cell lines,indicating the requirement of Wnt signaling.

FIG. 10 depicts exemplary dominantly active β-catenin (DA-cat) elicitingTOPFLASH activity in mouse tumor-derived cells (line 1029) and isrepressed by dominant negative effector, TCF4 (DNTCF4).

FIG. 11 depicts exemplary NMuMG cells that were transfected with Wnt7a,DKK3a and DKK3b and grown for 48 hr. Cell lysates were prepared intriplicate and luciferase expression determined using standardlaboratory methods.

FIG. 12 depicts exemplary C8DKK3b^(CFPP+/−) cells grown for 72 h withthe methylase inhibitor 5′ azacytidine.

FIG. 13 depicts exemplary, A. Domain organization of the TAT-DKK3bfusion protein. B. Effects of TAT-DKK3b on b-catenin dependent geneexpression in Wnt-treated HEK293T or NMuMG cells. Cell were transfectedwith plasmid cocktails containing Wnt, promoter-luciferase, and atransfection control, pRhluc. Cells were exposed to TAT-DKK3b for 5 minand grown for 24 h; lysates were made and luciferase activitydetermined.

FIG. 14 depicts exemplary effects of TAT-DKK3b on cell proliferation andmetastasis.

FIG. 15 depicts exemplary pTAT/pTAT-HA construct.

FIG. 16 depicts exemplary optimized PTD Plus strand; Optimized PTD minusstrand; Annealed PTD domain from synthesized primers.

FIG. 17 depicts exemplary map of N-terminus of the “optimal” PTD-DKK3bfusion protein.

FIG. 18 depicts exemplary original TAT-HA Construct.

FIG. 19 depicts exemplary optimized TAT.

FIG. 20 depicts exemplary optimized PTD Construct.

FIG. 21 depicts exemplary primers for in-frame cloning of DKK3B (mouseand human).

FIG. 22 depicts exemplary gel-purified pRSETA-hDKK3b.

SUMMARY OF THE INVENTION

The invention provides novel therapeutic approaches to cancer treatmentthat exploits tumor suppressor functions of DKK3b. A novel tumorsuppressor has been identified that originates from an internaltranscription start site of the Dkk3 gene locus that harbors all of theanti-cancer properties. Rather than the current therapeutic efforts thatare all directed at an artifactual ER stress response due to exogenousexpression of the full length secreted and glycosylated DKK3 geneproduct, the present invention is promised to bring a unique approachthat changes the direction of the field.

In one aspect, the invention generally relates to the identified humanDKK3b protein.

In another aspect, the invention generally relates to a recombinantvirus genetically modified to express human DKK3b protein.

In yet another aspect, the invention generally relates to an isolatednucleic acid molecule comprising a polynucleotide sequence that encodesDKK3b protein.

In yet another aspect, the invention generally relates to a recombinanttransgene comprising a polynucleotide that encodes DKK3b protein.

In yet another aspect, the invention generally relates to an isolatedrecombinant human DKK3b protein.

In yet another aspect, the invention generally relates to host celltransformed with an isolated recombinant human DKK3b protein.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising a recombinant virus geneticallymodified to express human DKK3b protein and a pharmaceuticallyacceptable carrier.

In yet another aspect, the invention generally relates to a method fortreating cancer or inhibiting tumor progression in a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising a recombinant virus genetically modified toexpress human DKK3b protein and a pharmaceutically acceptable carrier.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising human DKK3b protein and apharmaceutically acceptable carrier.

In yet another aspect, the invention generally relates to a method fortreating cancer or inhibiting tumor progression in a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising DKK3b protein.

In yet another aspect, the invention generally relates to a method forinducing a tumor suppression effect in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising DKK3b protein.

Cancer that may be therapeutically treated according to the disclosedinvention can be any type of cancer, including carcinoma, lymphoma,blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma,schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoidmalignancy, squamous cell cancer, epithelial squamous cell cancer, lungcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer, atumor of the biliary tract, and head and neck cancer.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel therapeutic approaches to cancertreatment that exploit the tumor suppressor functions of DKK3b. Moreparticularly, a novel tumor suppressor has been identified thatoriginates from an internal transcription start site of the Dkk3 genelocus and this gene product harbors all of the anti-cancer properties ofthis gene. Rather than the current therapeutic efforts that are alldirected at an artifactual ER stress response due to exogenousexpression of the full length secreted and glycosylated DKK3 geneproduct, the present invention is promised to bring a unique approachthat will change the direction of the field.

Unchecked signaling by the Wnt pathway signaling molecule, β-catenin, iscommon in cancer and the Dickkopf-related protein 3 (DKK3) is one of themost promising tumor suppressor molecule(s) that controls β-cateninlevels. Tumor malignancy is inversely related to DKK3 levels andover-expression arrests cell proliferation in nearly all cancer cells.Even though DKK3 does not block receptor activation by Wnt, theanticancer actions of the secreted DKK3 glycoprotein are widely presumedto be due to inhibition of the Wnt receptor complex. However, recentreports challenge this conventional view of DKK3 action. Yeasttwo-hybrid analysis revealed that DKK3 interacted with the ubiquitinligase, β-transducin repeats containing protein (βTrCP) and subsequentco-immunoprecipitation studies identified DKK3 as part of anintracellular complex with (βTrCP) and β-catenin that prevents β-cateninnuclear translocation. DKK3 was also shown to activate the N-terminalJun kinase (JNK) stress pathway leading to cancer cell loss. Thesefindings illustrate an important role for intracellular signaling byDKK3. We recently discovered that the Dkk3 locus encodes two geneproducts, a secreted glycoprotein (DKK3a) that does not impact Wntsignaling, and a novel, intracellular protein, DKK3b, which inhibits Wntsignaling. We propose that the intracellular DKK3b is responsible forthe published anti-cancer effects of this tumor suppressor and acts byblocking the nuclear import of β-catenin and increasing cell death, twofundamental cellular events that arrest tumor growth and propagation.

The Molecular Mechanism of DKK3b Action

There is general agreement that 1) Dkk3 expression levels are inverselyrelated to tumor malignancy, and that 2) DKK3 over-expression haltscancer cell proliferation. (Lodygin, et al. 2005 Cancer Res65(10):4218-4227; Veeck, et al. 2008 Breast Cancer Res 10(5):R82; Veeck,et al. 2012 Biochim Biophys Acta 1825(1):18-28; Yue, et al. 2008Carcinogenesis 29(1):84-92; Edamura, et al. 2007 Cancer Gene Ther14(9):765-772; Gu, et al. 2011 World J Gastroenterol 17(33):3810-3817;Medinger, et al. 2011 Thrombosis and haemostasis 105(1):72-80; Pei, etal. 2009 Virchows Arch 454(6):639-646.) Despite these findings, themechanism(s) of DKK3 action remain completely unknown. Unlike other DKKfamily members, DKK3 does not bind to the Wnt receptor complex. (Niehrs,et al. 2006 Oncogene 25(57):7469-7481; Barrantes, et al. 2006 Mol CellBiol 26(6):2317-2326.) Moreover, deletion of mouse Dkk3 gene produced abenign phenotype with no increase in tumor formation.

Our discovery of a novel intracellular DKK3 gene product, DKK3b(previously named D2p29), produced from transcripts originating from aninternal start site, redefines and reconciles these disparateobservations. (Leonard, et al. 2000 J Biol Chem 275(33):25194-25201.)DKK3b possesses all the anti-tumor effects previously ascribed to DKK3a.In fact, the targeting strategy used to delete the mouse Dkk3 ablatedonly DKK3a, leaving DKK3b untouched, thus explaining the lack ofincreased tumorigenesis. (Barrantes, et al. 2006 Mol Cell Biol26(6):2317-2326.)

Recent studies show that DKK3 specifically binds to the E3 ubiquitinligase component, β-transducin repeat containing protein (βTrCP), andthat this complex in turn binds unphosphorylated β-catenin preventingits nuclear import. (Lee, et al. 2009 Intl Cancer 124(2):287-297; Hoang,et al. 2004 Cancer Res 64(8):2734-2739.) Since the membrane barriermakes it improbable that the secreted DKK3a glycoprotein acts inside thecell by direct association with E3 ubiquitin ligase machinery, the novelDKK3b gene product becomes the likely tumor suppressor responsible forattenuating Wnt signaling. Understanding the details of how DKK3barrests tumor growth offers a real opportunity to develop effectivetherapeutics that defeat oncogenesis at its source.

The present invention defines the molecular mechanism(s) of DKK3b tumorsuppressor function using both prostate and breast cancer cells asmodels for cancers in general and addresses the anti-tumor effects ofsite-specific delivery of DKK3b. By re-directing the focus of work tothe authentic important tumor suppressor, the invention establishes animportant foundation for rational therapeutic approaches to cancertreatment.

In silico analysis revealed that DKK3b was encoded by exons 3-8 of theDkk3 gene, and the presence of this novel DKK3 gene product in brainsfrom the Dkk3^(−/−) mouse provided insight into its origins. (Barrantes,et al. 2006 Mol Cell Biol 26(6):2317-2326.) Deletion of exon 2 from themouse Dkk3 locus (FIG. 1a ) clearly eliminated expression of DKK3a, butdoes not affect DKK3b transcripts starting at exon 3 (FIG. 1b ) and ledto a nearly 3-fold increase in the ˜30 kDa DKK3b protein (FIG. 1c ).Promoter activity was localized to ˜250 bases upstream of exon 3, anddeletion of the TATA box blocked promoter function. ChIP analysis showedthat the DKK3b promoter was functional in vivo (FIG. 1d, 1e ). QPCRusing exon 2 and exon 3 specific DKK3 primer sets found that the DKK3atranscript (exons 2-8) accounted for 50-55% of the mRNA in primarycells, while the DKK3b transcript (exons 3-8) contributed ˜45% of thetotal. These data show that the Dkk3 gene encodes two functionaltranscripts: one encoding a ˜60 kDa secreted glycoprotein DKK3a; and asecond 30 kDa intracellular protein, DKK3b.

To evaluate the tumor suppressor function of the two DKK3 gene products,DKK3a and DKK3b were cloned into the Tet-inducible, expression vector(pTRex) and transfected into PC3 prostate tumor cells thatconstitutively express the Tet repressor protein. The data in FIG. 2show that DKK3a does not slow cell growth. On the other hand, DKK3bcompletely arrests cell proliferation, and leads to the loss of PC3cells at later time points. These data illustrate that DKK3a lacks thetumor suppressor function associated with its intracellular form, DKK3b.

Characterization of the Molecular Events Mediating DKK3b-DependentGrowth Arrest

The ability of DKK3b to arrest cancer cell proliferation could be due tospecific cell cycle arrest, enhanced apoptosis, and/or induced cellsenescence. To explore these cellular event(s) without the confoundingimpact of aneuploidy/polyploidy and the genetic instability that areinherent in tumor cell lines, initial studies are done in immortal C8astrocytes. These cells have a stable 2N gene copy number and show allof the properties of a primary astrocyte. They are easily transfectedand have marginal native DKK3b. Epitope-tagged DKK3b are re-expressed inC8 cells using the tetracycline (tet)-inducible pTRex expression systemthat can be titrated to generate cells harboring increasing quantitiesof the tumor suppressor as illustrated in DU145 cells in FIG. 3.

Since we propose that one of the primary actions of DKK3b is initiatedby stopping the nuclear import of β-catenin, a TCF-driven TOPFlashluciferase reporter are used to monitor βcatenin dependent geneexpression. MnuMG Wnt-dependent breast cancer cells were transfectedwith DKK3a and DKK3b and the TCF-driven luciferase (pTOPFlash), and theeffects of the two DKK3 gene products on basal and Wnt7a stimulatedreporter activity determined. As shown in FIG. 4, Wnt stimulationresulted in a 10-14 fold increase in TOPFlash signaling in the absenceof any DKK3 or in the presence of the inactive DKK3a. Expression ofDKK3b decreased TOPFlash signaling to near basal levels. These dataillustrate the specific ability of DKK3b to silence β-catenin signaling,a central thesis of the molecular basis for the action of this tumorsuppressor.

To confirm the direct interactions between βTrCP and DKK3b,RNAi-mediated knockdown of βTrCP in C8 pTOPFlash reporter cells is usedto explore the impact of altered βTrCP expression on the ability ofDKK3b to suppress TCF-reporter expression. Controls include thescrambled RNAi pools and/or off-target shRNAi lentivirus and theefficiency of knockdown are measured by immunoblot andimmunocytochemistry. The loss of βTrCP will eliminate theDKK3b-dependent suppression of TCF-TOPflash reporter activity.

Based on these initial studies, the influence of Dkk3 derived geneproducts on cell proliferation and the cell cycle are examined in LNCaP,PC3 and DU145 cells using the tetinducible DKK3a and DKK3b constructs.Cell proliferation (WST-1), viable cell counting, and cell cycleanalysis using fluorescent activated cell sorting (FACS) of BrdU/7-AADlabeled cells are done. As shown in Table 1, DKK3b arrests the humanprostate cancer PC3 cell at the G0/G1 phase of the cell cycle, whileDKK3a had no effect on the cell cycle or on cell proliferation (also seeFIG. 2).

TABLE 1 Effects of DKK3a and DKK3b on cell cycle in PC3 cells Cell CycleAnalysis (% total cells) G0/G1 S G2/M control 44.1 27.2 17.1 DKK3a 42.328.5 16.3 DKK3b 95.7 2.6 1.2

Because DKK3b led to both growth arrest and loss of PC3 cells (FIG. 2),the impact of DKK3b on apoptosis is evaluated using flow cytometry,TUNEL assays, caspase-3 analysis and annexin V staining. TUNEL,caspase-3 measurements, and cell proliferation using Ki67 are repeatedusing standard immunocytochemical approaches to provide confirmation ofthe effects of DKK3b on apoptosis determined by flow analysis.

Once these basic functional parameters are established, the role of theDKK3b:βTrCP complex in tumor cell growth arrest are examined using theRNAi knockdown of βTrCP strategy detailed above for C8 cells. Loss ofβTrCP in cancer cells will thwart DKK3b-induced growth arrest. Theresults of these studies will establish the role of DKK3b:βTrCP complexin modulating the β-catenin proliferation signal. DKK3b may affect morethan one cellular pathway; however, focusing on the specificcharacterization of the inhibitory role of a DKK3b:βTrCP complex on cellgrowth provides a clear link between the well-known impact of β-cateninon cell proliferation and the tumor suppressor function of DKK3b.

Identification of DKK3b Modulated Signaling Pathway(s)

We propose that the DKK3b:βTrCP complex suppresses TCF-driven geneexpression by preventing β-catenin from reaching its nuclear TCF target.Thus, analysis of the more than 50 known TCF-driven genes provides awell-defined end point to evaluate the efficacy of the DKK3b:βTrCPcomplex. (Willert, et al. 2002 BMC developmental biology 2:8.) RNA-seqprovides an unbiased approach that allows transcriptome analysis of thewhole set of TCF target genes. This contemporary genomics methodologyprovides a data set that not only allows the known TCF-driven genes tobe evaluated, but can also be mined for unanticipated DKK3b dependentchanges in the transcriptome. RNA-seq is a significantly morecost-effective and a less biased approach than commercially availablePCR based array sets when cost/data point is considered. To determinethe impact of DKK3b on TCF-driven gene expression, RNA-seq is used todefine the impact of the inhibitory DKK3b:βTrCP complex on expression ofa known cohort of ˜110 TCF-driven gene targets.

From the RNA-seq analyses, 10-15 gene products are selected and used toassemble a DKK3b signaling array (DKK3b Response Array, DK3RA) toreliably report DKK3b function in prostate and breast cancer cells.Changes in the cellular content of these target protein(s) are confirmedusing multiplexed, Millipore EpiQuant luminex assays of whole celllysates, immunoblot analysis or immunocytochemistry. These studies aredesigned to demonstrate that the DKK3b tumor suppressor inhibitsβ-catenin signaling and provides a validated set of DKK3b alteredtranscripts (DK3RA) that can be used to monitor tumor suppressorfunction. Additionally, an invaluable data repository of DKK3b alteredtranscripts that can be mined for novel signaling partners will begenerated and archived.

The Role of DKK3b: fiTrCP Interactions on the Subcellular Redistributionof β-Catenin

Over-expression of DKK3b can initiate the redistribution of β-cateninfrom the cytoplasm to the cell membrane without altering total β-cateninlevels. (Hoang, et al. 2004 Cancer Res 64(8):2734-2739.) Coupled withthe finding that an intracellular DKK3b:βTrCP complex bindsunphosphorylated β-catenin and can lead to β-catenin degradation, it islikely that the DKK3b:βTrCP complex performs at least three roles: 1)β-catenin sequestration; 2)β-catenin relocation; and 3) β-catenindegradation. Since these roles are likely to be temporally related, weuse confocal, total internal reflectance fluorescence (TIRF) microscopyand real-time digital imaging to define the dynamics of subcellularredistribution of β-catenin, DKK3b and βTrCP in C8 cells and in ourcancer cells lines expressing tet-induced, increasing concentrations ofDKK3b. (Lee, et al. 2009 Int J Cancer 124(2):287-297.) Our prior workdetailing the dynamics of DKK3b trafficking guides our completion ofthese studies. (Farwell, et al. 1990 J Biol Chem 265(30):18546-18553;Stachelek, et al. 2000 J Biol Chem 275(41):31701-31707; Stachelek, etal. 2001 J Biol Chem 276(38):35652-35659.) In its simplest form, themembrane-bound DKK3b:βTrCP complex attracts unphosphorylated β-cateninleading to its membrane association. If adherens complexes are nearbythis complex may deposit β-catenin in these attachment structures.(Hartsock, et al. 2008 Biochim Biophys Acta 1778(3):660-669.) If not,the inactive complex may be degraded in the proteasome.

Characterization of the Organ/Tissue Distribution and ExpressionProfiles of DKK3b in the Mouse

Little is known about the tissue distribution, developmental timingand/or the expression profile of DKK3b, and traditionalimmunocytochemical approaches are impractical because availableantibodies recognize both DKK3b and DKK3a. To define the basicdevelopmental timing and tissue distribution of DKK3b expression, we usethe zinc-finger nuclease (ZFN)-based gene editing approach to produce aDkk3b reporter mouse by redirecting the DKK3b promoter to driveexpression of a fluorescent reporter (FIG. 4). (Clark, et al. 2011Zebrafish 8(3):147-149; Collin, et al. 2011 Stem Cells 29(7):1021-1033;Kim, et al. 2009 Genome research 19(7):1279-1288.) During validation ofthe ZFN pairs, gene-edited, Dkk3b-promoter driven CFP C8 (C8^(Dkk3bCFP))cells are generated by default that are a valuable cell based reporterfor high-throughput screening of combinatorial libraries.

Systematic functional analysis of the Dkk3b promoter is performed usingCFP expression as the endpoint and in silico analysis (TranFac,Genomatrix algorithms) is used to identify individual Dkk3b promoterelements. This promoter survey approach will identify one or morepromoter modulators that modulate transcription from this locus.

The Dkk3b reporter mouse provides two important in vivo models. 1)Heterozygotes (Dkk3b^(+/CFP)) express all three gene products from theDkk3 locus, DKK3a, DKK3b and CFP. These mice are used in define thedevelopmental timing and tissue distribution of DKK3b in embryos,neonates and adults. DKK3b promoter driven CFP is expected to showwidespread distribution in tissues. 2) Homozygotes (Dkk3b^(CFP/CFP)) arefunctional knockouts that express DKK3a, but not DKK3b, because theDkk3b-promoter of both alleles has been diverted to drive CFPexpression. These Dkk3b “knockout” mice, unlike the original Dkk3aknockout, lose the DKK3b tumor suppressor and are expected to showmarkedly increased cancer risk and potential developmentalabnormalities.

Methods and Procedures

Biological Assays for Cell Proliferation, Cell Cycle, Apoptosis andSenescence

Cell proliferation is monitored using the WST-1 reagent according tomanufacturer's instructions. Cell proliferation, cell cycle analysis,and apoptosis are conducted using FACS analysis of BrdU tagged cellsthat are post labeled with fluorescent antibodies directed againstepitope-tagged DKK3b and Annexin V (apoptosis). Alternatively, apoptosisare followed by TUNEL assays according to established methods in thelaboratory. Cell senescence is determined by β-galactosidase activityusing commercially available kits. All analyses are done in triplicateand repeated at least three times to validate the data.

DKK3 cDNA Constructs, Plasmid, Viral Expression Constructs andImmunological Probes

GATEWAY technology (Invitrogen) is used to generate entry plasmidsharboring epitope tagged DKK3a and DKK3b. This shuttle vector systempermits the rapid assembly of a wide repertoire of plasmid or viralexpression vectors by phage-based recombination. Both constitutive andtet-inducible destination vectors are used in the laboratory. GATEWAYtechnology is also available to shuttle PCR generated shRNAi into cellsor tissues. A complete collection of retroviral and lentiviral basedshRNAi human and mouse libraries are available.

Deep Sequencing of DKK3b Altered RNA Libraries

Cells harboring tet-inducible DKK3a or DKK3b are exposed to tetracyclineand grown for up to 24 h. Total RNA are isolated by Qiagen RNeasycolumns and poly A+ mRNA isolated by oligo-dT purification. TriplicatemRNA-seq libraries from control (no tet), DKK3a and DKK3b expressingcells are made using commercially available kits.

Gene Editing of the Dkk3b Promoter Locus

Dkk3b promoter targeted ZFN pair selection is guided by methodsdeveloped by Wolfe. (Wolfe, et al. 2003 Biochemistry 42(46):13401-13409;Meng, et al. 2008 Nature biotechnology 26(6):695-701; Zhu, et al. 2011Development 138(20):4555-4564.) cDNAs encoding target ZFN pairs aresynthesized de novo and cloned into the pCS-Fok1 vector. ZFN targetmodification is done in the isogenic C8 cell line and target specificityevaluated using PCR. Validation of ZFN target modification are done byCel-1 assays, by single-stranded oligo homologous recombination (HR)repair and by HR-mediated insertion of a floxed CFP reporter. (Chen, etal. 2011 Nature methods 8(9):753-755.) Off-target ZFN activity areevaluated in silico and the top 3 off-target sites interrogated by PCRanalysis. Validated ZFN pair(s) that edit the target region of the Dkk3bpromoter and the HR based CFP-pA cDNA construct are used to make Dkk3breporter mice.

In vitro synthesized ZFN mRNAs and the Dkk3b-promoter targetedHR-pLox-CFP-pApLox repair cDNA are injected into 50-75 fertilizedC57BL/6 mouse oocytes in the Transgenic Animal Modeling Core at UMMS.Single cell embryos are implanted into surrogates and offspring aregenotyped using Dkk3b-promoter specific PCR. Dkk3b-promoter gene editedmice are expanded into two colonies: Dkk3b^(+/CFP) heterozygotes arebred for use in developmental and tissue distribution studies, andDkk3b^(CFP/CFP) homozygotes are bred for evaluation of tumorsusceptibility of the Dkk3b knockout mouse. If the loss of Dkk3b proveslethal, the basis for lethality is then determined and thetissue-specific Cre expression is used to excise the CFP reporter andrestore Dkk3b expression in the tissue(s) required for survival.

With identification and characterization of the impact of a DKK3b:βTrCPcomplex on the biology of β-catenin signaling and the tumor suppressorfunction of DKK3b, the ability of DKK3b to “sequester” β-catenin, arresttumor cell growth and block TCF-driven proliferation/survival signalsprovides a straightforward mechanism for tumor suppressor function. Theunbiased, RNA-seq, bioinformatics-based approach is used to identify theaffected target gene pathways. From these data, a validated set of DKK3breporter genes will be selected (DK3RA) and used to monitor DKK3bfunction. Analysis of the dynamics of DKK3b-induced shuttling ofβ-catenin between different intracellular compartments is astraightforward cell biology problem that is approached by static anddynamic imaging paradigms. These data provide basic information on whatsteps in the process can be targeted for therapeutic intervention.

Additionally, generation of the DKK3b^(+/CFP) reporter cell line andDKK3b^(+/CFP) mouse models are invaluable for the study oftumorigenesis. Contemporary gene editing strategies using ZFN technologysignificantly reduce the time required to develop testable biologicalmodels. The C8^(Dkk3bCFP) cells generated during the validation processprovide a significant value-added benefit—they are useful to exploremethods of Dkk3b promoter activation and for the discovery of smallmolecules that alter DKK3b expression. Production of the Dkk3b reportermouse is significantly more cost effective than traditional ES cellbased homologous recombination strategies and decrease the time togenerate a viable mouse model by >80%. Characterization of Dkk3bpromoter function can be directly applied to in vivo intervention of theoncogenic process and to develop novel therapeutic approaches to cancer.

Mechanistic Analysis of DKK3b-Induced JNK Activation in Prostate Tumors

The JNK pathway is widely recognized as an important regulator of tumorbiology and serves as a key cellular defense mechanism to preventgenetic instability and the development of aneuploidy. (Kennedy, et al.2003 Cell Cycle 2(3):199-201; Vivanco, et al. 2007 Cancer Cell11(6):555-569; Whitmarsh, et al. 2007 Oncogene 26(22):3172-3184;MacCorkle, et al. 2004 J Biol Chem 279(38):40112-40121;Miyamoto-Yamasaki, et al. 2007 Cell Biol Int.; 31(12):1501-1506; Nakaya,et al. 2009 Cell Biochem Funct 27(7):468-472; Wang, et al. 2009 J Pathol218(1):95-103.) Previous reports have shown that DKK3 over-expression intumor cells increases JNK activity leading to inhibition of cellproliferation and increased cell loss mediated, in part, by apoptosis.(Edamura, et al. 2007 Cancer Gene Ther 14(9):765-772; Abarzua, et al.2005 Cancer Res 65(21):9617-9622; Ikezoe, et al. 2004 Br J Cancer90(10):2017-2024; Kawano, et al. 2006 Oncogene 25(49):6528-6537.)

In our hands, DKK3b-dependent growth arrest in DU145 prostate tumor cellrequires, in part, the JNK stress kinase pathway (FIG. 5A). While DKK3bhalted cell proliferation, TATJBD inhibition of JNK activity blockedcell loss (FIG. 5A), suggesting that the JNK pathway is required forcell loss. These results agree with our observations that Jnk inhibitsprostate tumor formation in the well-established conditional Pten mousemodel of prostate cancer. PTEN (phosphatase and Tensin homology) is atumor suppressor commonly mutated in cancer that recently was shown tohave a direct role in preventing chromosomal instability: ΔPten/ΔJnkmice lack PTEN and JNK in prostate epithelium and develop moreaggressive cancer than animals lacking PTEN alone (ΔPten) (FIG. 5C).(Baker 2007 Cell 128(1):25-28; Blanco-Aparicio, et al. 2007Carcinogenesis 28(7):1379-1386; Trotman, et al. 2007 Cell128(1):141-156; Yin, et al. 2008 Oncogene 27(41):5443-5453; Shen, et al.2007 Cell 128(1):157-170; Puc, et al. 2005 Cancer Cell; 7(2):193-204;Wang, et al. 2003 Cancer Cell 4(3):209-221.) While this result clearlyillustrates the role for the JNK pathway in preventing this cancer, thebiological processes that JNK regulates remain unclear. Control ofgenetic instability represents a likely mechanism for JNK function toprevent cancer. Preliminary studies using murine embryonic fibroblasts(MEF) show that loss of JNK expression dramatically increaseschromosomal instability and led to aneuploidy (FIG. 5B).

The increased genetic instability resulting from the loss of JNK isresponsible for the more aggressive tumors in prostate epithelium (FIG.5C). We propose that DKK3b inhibits tumor growth, in part, by aJNK-dependent manner in vivo. The invention provides the firstmechanistic framework for understanding how the DKK3b-JNK signaling axisprevents tumorigenesis and thus significantly advances our understandingof the role of this kinase pathway in human cancer. Thus, the inventiondemonstrates a role for JNK in preventing the genetic instability thatpromotes tumor formation and defines the molecular connections betweenDKK3b and the JNK pathway.

Characterization of the DKK3b-JNK Signaling Axis in Prostate Cancer

Expression of DKK3 is lost in most prostate tumors, and DKK3b is thebiologically relevant tumor suppressor from the Dkk3 locus. The JNKactivating kinase Mkk4 is also mutated in a subset of primary humanprostate tumors. (Whitmarsh, et al. 2007 Oncogene 26(22):31723184; Kim,et al. 2001 Cancer Res; 61(7):2833-2837; Taylor, et al. 2008 Cancer Lett272(1):1222.) However, it is unknown if loss of both gene productssynergize to generate a more severe cancer. The invention establishescorrelative and causative connections between DKK3b and the JNK pathwaythat demonstrate the importance of this signaling axis in preventingprostate cancer. Primary human prostate tumor samples representing arange of Gleason scores for expression of DKK3b, DKK3a, β-catenin, MKK4,MKK7 and JNK and phospho-analogs are screened by immunocytochemistry,qPCR, and immunoblot analysis. Expression of the tumor suppressor PTENis also screened to determine if the connections between the DKK3b andJNK pathways require Pten mutations. We propose that tumor severity isinversely related to DKK3b levels and directly related to β-cateninnuclear localization, and the expression or activity of JNK, MKK4 and/orMKK7 is expected to be inversely correlated with tumor severity.

Analysis of Genetic Instability in Prostate Epithelium

Genetic instability is associated with DNA breaks, translocations andthe development of aneuploidy. The degree of genetic instability isdetermined in tissue sections of prostates from ΔPten and ΔPten/ΔJnkmice collected at several stages of tumor development including whenonly PIN lesions are evident. The enhanced prostate tumorigenesis inΔPten/ΔJnk mice indicates that regulatory mechanisms that preventuncontrolled cell growth (such as apoptosis or senescence) are defectivedue to loss of JNK. Loss of these pathways disconnects the DKK3b tumorsuppressor from the genomic surveillance machinery and ultimately leadsto more aggressive tumor formation.

Tumor Inhibition Using Lentivirus Delivery of Activated JNK or DKK3b

The idea is tested that prostate-specific expression of DKK3b oractivated JNK inhibits PIN formation and tumor growth in murine modelsof prostate cancer. For these studies, murine models are modified toinclude a prostate-specific luciferase reporter to visualize tumorgrowth in living mice. Lentiviral delivery system is used to expressDKK3b and/or a constitutively active version of JNK specifically underthe control of the prostate-specific probasin promoter. (Vivanco, et al.2007 Cancer Cell 11(6):555-569.) The effect of prostate-specificexpression of DKK3b or active JNK on tumor growth is monitored over timein live animals by evaluating expression of the luciferase reporter.

Dependence of JNK for Anti-Tumor Effects of DKK3b

Although DKK3b blocks cancer cell proliferation ex vivo, through amechanism that involves JNK, it is unknown if this holds true in vivo.Gene replacement by lentiviral constructs will be used to express bothDKK3b and TAT-JBD—a specific peptide inhibitor of JNK—in prostate tumorsof ΔPten/Luciferase reporter mice. The effect of JNK inhibition on DKK3bfunction is evaluated by analyzing PIN formation in sections ofprostates from sacrificed mice and monitoring tumor regression over timein live animals. We expect that loss of JNK activity will lead to morePIN formation.

Molecular Mechanism(s) of JNK Activation by DKK3b

The activation of upstream kinases known to lead to JNK phosphorylationis examined. The RNA-Seq data set is queried to identify changes in geneexpression affecting pathways known to regulate JNK signaling. MultiplexELISA is used to analyze changes in cytokine expression profiles thatcould lead to JNK activation. The salient findings from our in vitroanalysis are confirmed in vivo using gene replacement/RNAi knockdown ofcandidate pathway members suspected of playing a role in tumorregression and/or suppression of PIN formation. For example,identification in vitro of a DKK3b-altered MAPKKK or DKK3b-dependentproduction of JNK-activating cytokine(s) will be experimentallyevaluated in tumors expressing DKK3b.

Methods and Procedures

Laser Scanning Cytometry

Laser scanning cytometry is used to analyze ploidy changes in prostatetumor cells from ΔPten and ΔPten/ΔJnk mice. Because this method usessections of intact prostate, tissue architecture is preserved allowingfor the assessment of PIN formation. Prostate tissue sections arestained with antibodies to E-cadherin (to mark stromal cells),N-cadherin/cadherin-11 (to mark tumor cells) and propidium iodide tolabel DNA followed by analysis using laser scanning cytometry. Analysisof DNA content in stromal cells (positive for E-cadherin but negativefor Ncadherin and cadherin-11) serves as an internal control for thediploid cell population. The DNA content of tumor cells (positive forN-cadherin and cadherin-11 and low levels of E-cadherin) is compared tostromal cells to determine ploidy changes. Control experiments includeprostates from wild type, Pb-Cre, Pten^(f/f) and Jnk1^(f/f)/Jnk2^(−/−)mice.

Morphological Methods

Standard immunohistochemical and immunofluorescent techniques is used toanalyze prostate tissue morphology and the presence of DNA strand breaksin ΔPten/ΔJnk mice. Prostate tissue sections are stained withhematoxylin and eosin to reveal morphology. DNA strand breaks isassessed using antibodies to phospho-H2Ax and p53BP. Chromosomalrearrangements are evaluated using the spectral karyotyping (SKY)technique. Cellular senescence is determined by assaying the senescencemarker β-galactosidase. Apoptosis is evaluated by assessing the levelsof nicked DNA using the (TUNEL) assay and caspase-3 activity.Proliferation is quantified using Ki67 or BrdU immunostaining. Prostatetissue from wild type, Pb-Cre, Pten^(f/f) and Jnk1^(f/f)/Jnk2^(−/−) miceserve as controls. Tumor sections will be evaluated using confocalmicroscopy and fluorescent images analyzed for statistical differencesusing Image Pro Plus® software.

Analysis of Human Prostate Tumor Gene Expression

Tumor specimens are obtained from the UMMS Tissue Bank and representvarious grades of tumor severity based on Gleason scores. RNA isisolated from tumor samples and screened for expression of DKK3b, DKK3a,β-catenin, Pten, Mkk4, Mkk7 and Jnk using qPCR assays and resultsconfirmed using immunoblot and/or immunocytochemistry to detect proteinexpression. These experiments establish, for the first time, acorrelative connection between DKK3b, the JNK pathway, and prostatecancer severity. We expect an increasing Gleason score to be positivelycorrelated to the loss of both Dkk3b and Mkk4 gene expression.Alternatively, mutational inactivation could functionally suppress DKK3band MKK4 activities. Therefore, the Dkk3 and Mkk4/7 genes are sequencedto identify possible inactivating mutations that do not affect overallexpression.

Mouse Models of Prostate Cancer

Our novel murine models of prostate cancer are crossed to a prostatespecific luciferase reporter mouse strain and the progeny are used forthese studies. Prostate specific luciferase expression is directed by atransgene that expresses the firefly luciferase gene under the controlof a probasin promoter modified to include two androgen responseelements (ARR2-Pb-Lux, (Ellwood-Yen, et al. 2006 Cancer Res66(21):10513-10516), hereafter as Lux). Lux reporter mice are crossed toour conditional mice that lack expression of PTEN (ΔPten) or PTEN andJNK (ΔPten/ΔJnk) in the prostate, generating both Lux-ΔPten andLux-ΔPten/ΔJnk strains for live animal imaging studies.

Construction and Use of Prostate-Specific, Lentiviral Delivery System

Lentiviral constructs for prostate-specific expression of JNK,constitutively active JNK (a fusion between the upstream JNK activatorMKK7 and JNK, MKK7-JNK), a specific JNK inhibitor TAT-JBD (a fusionprotein of the JNK Binding Domain (JBD) of the JIP1 scaffolding proteinappended to the HIV TAT sequence) and DKK3b are constructed as detailedherein. Expression of all constructs is driven by the ARR2-Pb promoterto ensure prostate specific expression and include an epitope tag tofacilitate detection of expressed proteins. Lentiviral supernatants areadministered to live animals either by tail vein injection oralternatively, by direct intratumoral injection. Preliminary controlexperiments using wild-type mice are done to optimize the percentage ofprostate cells infected through either mode of injection usingimmunofluorescent microscopic analysis of prostate sections stained withepitope specific antibodies.

Quantitation of Tumor Growth in Live Animals

The Lux-ΔPten and Lux-ΔPten/ΔJnk mouse strains develop prostate tumorsthat express the firefly luciferase reporter gene, allowing us tomonitor tumorigenesis in live animals. Sedated mice are injected withthe luciferase substrate, luciferin, and emitted light are measuredusing the Xenogen® IVIS imaging system. Both early stage primary lesionsand metastases that have spread to other organs can be visualized usingthis system. Wild type Pb-Lux mice that lack tumors are used as negativecontrols. Specific gene replaced mice (DKK3b, MKK7-JNK and TAT-JBD) areimaged before viral infection to establish a baseline for tumor size.Subsequent images are collected weekly for 4 weeks to determine theeffect(s) of modulation of the DKK3b and JNK pathways on tumor growth.Initial control studies are done to optimize the time between luciferininjections and live animal imaging. Additional control studies includeinjection of an empty lentiviral construct.

Lentivirus Production and Delivery

Production and validation of concentrated lentiviral supernatants aredone using standard protocols established in our laboratories. Viraltiters are determined by limiting dilution and expression of thespecific epitope-tagged proteins will be determined byimmunofluorescence techniques. Administration of lentiviral particlesare achieved by injection into the tail vein of mice twice over twodays. Alternatively, direct delivery of lentiviral particles to theprostate of anesthetized mice are done through a small incision in theperitoneum in the lower abdomen. Approximately 10-20 μl of concentratedlentiviral stock (˜108 particles) are injected into the prostate and thewound closed using surgical sutures. Surgical recovery of the animals iscarefully monitored for signs of infection or other complications.Controls include animals injected with PBS or empty lentivirons.

Assessment of Prostatic Intraepithelial Neoplasia (PIN) Lesions

Standard histochemical and immunological staining techniques areemployed to evaluate PIN lesion development in our murine models ofprostate cancer. The prostate are harvested at time points spanning theonset of disease to the beginning of tumor formation. Serial sections ofprostate tissue are prepared from paraffin-embedded or frozen tissueblocks. One section are stained with hematoxylin and eosin to identifythe morphological disruptions that characterize PIN lesions and adjacentsections are used for immunological detection of lentiviral deliveredgene products using epitope tagged antibodies. The total number of PINlesions and the percentage of PIN lesions (if any) that expresslentiviral delivered gene construct(s) are determined from at least fivemice at every time point.

Endpoint Analysis of Prostate Tissue

For all experiments involving mice, prostate tissue are harvested at thetime of animal sacrifice. Paraformaldehyde-fixed prostate tissue isprocessed to generate paraffin and frozen tissue sections for analysisof PIN lesions, lentiviral-mediated gene expression and luciferaseactivity. Unfixed prostate tissue from additional mice is used fortranscript analysis using qPCR and for determination of proteinexpression by either immunoblotting or multiplexed ELISA (luminex)assays. The small size of prostates in mice that serves as negativecontrols are in early stages of disease require pooled tissue togenerate enough material for all analyses.

Statistics and Power Analysis

The number of mice in each group is determined by the statistical powerrequired to detect significant, biologically relevant differences. Ameaningful difference in means between groups can be detected usingt-test assuming normality. The difference in means is expressed in unitsof standard deviation (which accounts for the magnitude of inter-animalvariability). With 8-10 mice in each group, differences can be detectedbetween means that are greater than 1.25 SD (since smaller differenceswill not likely be biologically relevant) at a 0.05 significance level.We anticipate that increased Dkk3b expression will result instatistically significant tumor growth delay.

Most established tumors regardless of tissue origin show evidence ofgenetic instability. While it is likely that tumors from ΔPten/ΔJnk miceare genetically unstable, it is the analysis of the earliest events intumorigenesis—the formation of PIN lesions—that prove fruitful regardingcause and effect. To overcome analytical limitations due to the smallsize of early PIN lesions, we use laser scanning cytometry. A primaryadvantage of laser scanning cytometry is the retention of tissuearchitecture allowing for the identification of PIN lesions based onmorphology rather than tumor antigen(s) expression. This approach alsoovercomes the caveat that differences in cadherin expression may notoccur in early PIN lesions and/or that differential cadherin expressionmay require JNK-dependent AP-1-mediated transcription. Because tissuearchitecture is preserved in laser scanning cytometry, other criteriasuch as androgen receptor (AR) expression can be used to identify PINlesions: normal prostate tissue shows a well-defined expression of AR ina single layer of epithelial cells, whereas PIN lesions showdisorganized, multi-layer AR expression.

Analyzing DNA strand breaks in early PIN lesions as well as late stagetumors using immunocytochemisty and SKY, respectively, allows us toconnect increased DNA damage in PIN lesions to increased chromosomaltranslocations in late-stage tumors. While it is possible that we willnot find differences in genetic instability between ΔPten and ΔPten/ΔJnktumors on a per cell basis, the data obtained will provide valuableinformation because the frequency of appearance rather than the degreeof instability in affected cells may be the driving force that leads toincreased prostate tumor formation in ΔPten/ΔJnk animals. Such a resulthighlights the role of the JNK pathway in maintaining genetic integrity.

Another potential concern is that limitations/defects in upstreamactivators of JNK may prevent increased JNK expression from affectingtumor growth. To address this, we use the lentiviral delivery system toexpress a constitutively active MKK7-JNK fusion protein that bypassesthe need for upstream activating kinases. This serves an importantproof-of-concept test of our hypothesis that increasing JNK activitylevels inhibits tumor growth, and sets the stage for additionalexperiments that seek to increase endogenous JNK activity in tumors bymodulating the JNK pathway.

While we propose that DKK3b blocks cancer cell growth, in part, througha JNK dependent process, it is just as possible that DKK3b also worksthrough JNK-independent mechanisms to inhibit cancer cell growth. Thispossibility can be tested by expressing DKK3b in tumors of ΔPten/ΔJnkmice. If no change in tumor status occurs, then the protective functionof DKK3b requires JNK. Alternatively, DKK3b may induce a partialinhibition of tumor growth in the absence of JNK suggesting theexistence of alternative JNK-independent pathways that control tumorgrowth.

Finally, Lentiviral-mediated delivery of DKK3b and JNK to prostatetissue presents potential concerns. If tail vein delivery of lentiviralparticles does not attain levels of infection necessary to affect tumorgrowth using of virus, uninfected tumor cells will continue toproliferate and could, if present in sufficient numbers, mask any growthinhibition offered by DKK3b/JNK. This pitfall is of particular concernfor experiments designed to regress established tumors. To address thisproblem, pilot studies are performed using a GFP reporter virus todetermine if tail vein injection yields sufficient prostate infectionrates. If tail vein injection of viral particles proves inefficient,viral particles are injected directly into the prostate of anesthetizedmice through a small incision made in the abdomen. Initial controlstudies will determine volume/concentration of viral particles and thedegree of expression of exogenous proteins. Mice injected with PBS andempty lentiviral particles are run in parallel as additional controlsfor these experiments.

Relationship Between DKK3b and Triple Negative Breast Tumor Phenotypes

Like other epithelial cancers, DKK3b expression is altered in themammary tumors in our mouse model (FIG. 6), and shows a cell specific,inverse relationship with cytoplasmic βcatenin. This heterogeneity ofDKK3b and β-catenin expression was maintained in tumor-derived celllines indicating that different cohorts of tumor resident cells can beisolated and their molecular defect(s) characterized ex vivo (FIG. 7).

Loss of DKK3b may play an important role in the formation andmaintenance of so called “triple negative” breast cancers (TNBC). ER,PR, Her2 triple-negative breast cancers are a class of aggressive tumorswith few, if any, options for treatment. To examine this, two mousemodels are employed to explore the evolution of these tumors and themolecular details that contribute to their aggressive nature. The firstis our novel mouse model (TBP) that mimics TNBC due to the loss of threecritical tumor suppressor pathways: Rb; BRCA1; and p53 that are affectedin human TNBC. (Simin, et al. 2005 Cold Spring Haab Symp Quant Biol70:283-290; Kumar, et al. 2012 PLoS Genet. 8(11) e1003027; D'Amato, etal. 2011 PLoS One 7(9) e45684; Herschkowitz, et al. 2007 Genome Biol8(5):R76.) Our global gene expression survey of 13 mouse models revealedthat the TBP mouse mimicked human TNBC. The second model, MMTV-Wnt1, isa well-established model of breast cancer that also resembles TNBC, andwas exploited to study tumor-initiating cells. We hypothesize thatdys-regulated Wnt activity contributes to the tumor phenotype, andtargeting this pathway with DKK3b will block tumor progression, whichwill translate into significant clinical benefits for breast cancerpatients.

Since DKK3b suppresses tumorigenesis, in part, by limiting the functionof Wnt dependent tumor initiating cells and this is novel andsignificant. Our finding that DKK3b shows an inverse relationship withcytoplasmic β-catenin in mammary tumors is unprecedented and clearlyshifts the therapeutic paradigm to how DKK3b functions to arrest tumorcell growth. Characterization of the anti-tumor properties of DKK3boffers a novel therapeutic target with the promise for development of aneffective rational treatment of a lethal class of aggressive mammarytumors.

Evaluate the Contribution of DKK3b Expression to Mammary Tumorigenesis

The Wnt ligand is a potent morphogen and mitogen that fosters tumorprogression and the malignancy of TBP tumors at multiple stages ofdevelopment. The time course of both βcatenin and DKK3b expression aredefined in tissue sections from mammary tumors, pulmonary metastases,and MINs (mammary intraepithelial neoplasias) of TBP and Wnt1 mice.Normal patterns of β-catenin and DKK3b expression are assessed in wildtype control tissues. Tissue samples are examined usingimmunofluorescence, in situ hybridization, and multiplexed ELISA ofisolated tumor resident cells.

Cell sorting paradigms are used to enrich specific subsets of theheterogeneous tumor population using the cell surface markers CD49f,CD24 and CD61 that label tumor-initiating cells. Alternatively, lasercapture micro-dissection are used to isolate morphologically distinctcell populations from TNBC tumors. Cells are isolated by sorting ormicro-dissection and characterized by qPCR, multiplexed ELISA,immunoblot, and the DK3RA assay. A minimum of 24 TBP and 24 Wnt1 tumorsare systematically examined to establish the coincident or mutuallyexclusive expression of β-catenin and DKK3b. Our initial results showedhigh levels of DKK3b are associated with low levels of cytoplasmicβ-catenin within a single cell. The clinical relevance of these tumortargets is corroborated by evaluating patient-derived specimens. Tumorsare examined ranging in grade and clinical marker status, usingsufficient numbers to establish statistically significant associations,as we have published previously (46-49) to define the phenotype(s) ofdifferent tumor resident cells in TNBC tumors.

Examine the Effects of Altered DKK3b on Mammary Tumor Initiation

The effects of altered DKK3b expression on tumor initiation aredetermined using Wnt1-transformed, immortalized mouse mammary epithelialcells, NMuMG. DKK3a failed to alter proliferation of NMuMG cells, likePC3 and DU145 cells, while DKK3b slowed cell growth (FIG. 8). NMuMGcells expressing the tet-inducible DKK3b are transformed by Wnt1 and theeffects of increasing DKK3b on Wnt1-dependent tumor initiation evaluatedusing soft agar cultures and tumorsphere formation assays.

NMuMG cells do not form tumors unless transformed by oncogenic signalssuch as the Wnt ligand. Orthotopic transplants of tet-inducible,DKK3b-expressing NMuMG cells in NOD/SCID mice undergo transformation andthe effect(s) of DKK3b on tumorigenesis evaluated in vivo. Resultingtumors are harvested and DKK3b-dependent changes characterized bymorphology (H&E), proliferation (Ki67), apoptosis (TUNEL), β-catenindistribution patterns (total cell and cytoplasmic/nuclear ratio) andLEF/TCF-regulated target genes, such as ECadherin, N-Cadherin, AXIN2,Cyclin D1 and c-Myc. Since tumor-initiating cells are enriched by Wnt1stimulation, FACS (CD49f^(high), CD24^(low), CD61⁺) are used to isolateand quantify tumor initiating cell populations. (Cho, et al. 2008 StemCells 26(2):364-371; Vaillant, et al. 2008 Cancer Res 68(19):7711-7717.)We expect that increased levels of DKK3b will attenuate both tumorinitiation and tumor maintenance.

Establish the Impact of Endogenous DKK3b on Wnt1-Induced MammaryTumorigenesis In Vivo

The conditional, loss-of-function Dkk3b^(CFP/CFP) reporter mouse ismated with MMTV-Wnt1 mice (JAX). Importantly, the conditional deletionof Dkk3b can be reversed by coexpression of several mammary glanddirected (MMTV-, BLG-, WAP-) Cre recombinases. Our hypothesis is thatreduced DKK3b levels will aggravate Wnt1-induced tumor development dueto the unchecked increase of cytoplasmic β-catenin. Tumor latency andtumor phenotypes of the parent Dkk3b^(CFP/CFP) (Dkk3b knockout),MMTV-WNT1 (Dkk3b expressor), and crossed Dkk3b^(CFP/CFP)/MMTV-WNT1(Dkk3b knockout, tumor producers) and Dkk3b^(CFP/CFP)/MMTVWNT1/MMTV-Cre)(Dkk3b restoration, tumor producer) animals are compared. (Simin, et al.2005 Cold Spring Haab Symp Quant Biol 70:283-290; Herschkowitz, et al.2007 Genome Biol 8(5):R76; Lu, et al. 2011 Mol Cancer Res 9(4):430-439;Simin, et al. 2004 PLoS Biol 2(2):E22.) In the simplest analysis, weanticipate mice lacking DKK3b to have more tumors with faster onset, andthe Cre-repaired animals expressing DKK3b to have fewer tumors withslower onset.

Determine the Effects of Altered DKK3b on the Function of Mammary TumorInitiating Cells

Primary cells derived from mammary tumors in the TBP mouse withDKK3b^(high) (MMTVWnt1: Dkk3b^(+/CFP)) and DKK3b^(low)(MMTV-Wnt1:Dkk3b^(CFP/CFP)) expression patterns provide a uniqueresource to evaluate the effect of DKK3b activity on tumor-initiatingcells. Using FACS sorted pools of high and low DKK3b expressing cellsfrom primary tumors, the impact of DKK3b on tumor initiating capacitiesare examined by ex vivo and in vivo assays. We expect DKK3b high cellsto suppress Wnt signaling at the level of its intracellular transducer,βcatenin, and thereby reduce tumor initiation. In preliminary studies,we found that inhibition of the Wnt pathway markedly reduced tumorsphereformation in five independent tumor derived cell lines (FIG. 9).

The relationship between levels of DKK3b expression and the number ofcells showing the tumor initiating cell phenotype (CD49f^(high),CD24^(low), CD61⁺) in tumors are used to identify the impact of DKK3b ontumor-initiating cells from the TBP mouse. The DKK3b and βcatenin levelsin flow-sorted cells are assayed by qPCR and immunoblotting. We expecttumor initiating cells to have lower levels of DKK3b expression than theremaining cell population.

Next, we will establish the basal tumor initiating capacity of (CD49f⁺,CD44⁺, CD24⁻, EpCAM⁻) sorted primary human epithelial tumor cells. Cellswill be prepared from freshly harvested human tumor samples (UMass TumorBank) and in vitro (soft agar/tumorsphere) and in vivo (transplant)assays done as detailed above. Sorted cell populations will bemanipulated by viral delivery of Dkk3b cDNA (over-expression) or RNAi(knockdown) and the impact altered DKK3b expression on tumorigenesissystematically determined.

Demonstrate that Exogenous DKK3b Expression in Established Tumors CausesTumor Regression

Our murine model of transplanted cancer is modified to includeTBP-derived tumor cells that express TCF-driven TOPFlash, and atet-inducible DKK3b. These reporter cells allow us to visualize Wntstimulated tumor growth in a living mouse. DKK3b expression are inducedby oral, IP, or direct tumor injection of tetracycline at differentstages of tumor growth and tumor size is monitored by live animalimaging using the Xenogen® IVIS imaging system. Using this approach wecan directly relate DKK3b expression to pathway-specific changes intumor cell Wnt signaling and tumor regression. In vitro pilot studiesshow that Wnt-dependent luciferase expression was repressed by blockingTCF signaling (FIG. 10).

Methods and Procedures

Histology.

Standard immunohistochemical and immunofluorescent techniques are usedto analyze mammary tissue morphology as detailed herein.

FACS.

Markers of tumor initiating cells have been reported for mice(CD49f^(high), CD24^(low), CD61⁺) and human breast cancers (CD49f⁺,CD44⁻, CD24⁻, EpCAM⁻). All antibodies are commercially available.

Tumorsphere Assay.

Tumor cells are grown in non-adherent conditions (ultra low attachmentplates, Corning) in defined medium containing EGF and FGF. Mousetumorsphere forming units (TFU) are determined by counting. (Liu, et al.2007 Cancer research 67(18):86718681.) The time to generation and thetotal number of tumorspheres are the two parameters used as the assayreadout.

Cell Suspension and Transplantation.

To explore the ability of DKK3b to regress established tumors, a keyproperty of any therapeutic target, TBP cells harboring a TCF-drivenTOPFlash reporter are generated that express DKK3b or GFP (control)under control of a tetracycline-inducible promoter. Cells are injectedinto the mammary fat pads of syngeneic (FVB) recipient mice. A TBP cellline is also produced that also carries a stably integrated TOPFlashluciferase reporter (M50 Super 8×TOPFlash), driven by eight copies ofthe optimized TCFbinding element (FIG. 10).

For transplantation, 1×10⁶ cells are mixed 1:1 with Matrigel andinjected into the #2 mammary gland of recipient 7-8 week old FVB mice.Tetracycline is given by the drinking water, IP injection, direct tumorinjection, based on the optimal delivery method, when tumors reach 0.5cm in diameter as measured using calipers. Tumor growth is monitoredweekly by Xenogen® IVIS imaging system as detailed herein. When controltumors reach ˜1.5 cm in diameter, tumors will be excised and DKK3baffected signaling pathways characterized. Power calculations indicatedthat groups of 10-11 injected mice for each cell line are sufficient toachieve statistically significant results.

Statistics and Power Analysis.

Statistical methods and power analyses are described herein.

Interrupting Wnt signaling by reducing the fold-change of cytoplasmicβ-catenin has important translational implications as a novel regulatorypathway that can halt expansion of the mammary tumor-initiating cell.From this point of view, re-expression of DKK3b, either by in situactivation through its native promoter or by transfection of Dkk3b cDNAwill attenuate anchorage-independent growth of mammary epithelial cellsand block formation of tumorspheres, a property correlated with in vivotumor initiation capacity. (Liu, et al. 2007 Cancer research67(18):8671-8681.) The experiments described are straightforward indesign, validated by assay, and provide the experimental foundation forthe therapeutic application of DKK3b for tumor regression. In the invivo experiments, we expect loss of DKK3b to aggravate Wnt1tumorigenesis, but this requires inactivation of both alleles that mayhave untoward consequences. To avoid this, tumor specific DKK3bknockdown is done by tumor delivery of lentiviral siRNA, a means ofgenerating tissue targeted DKK3b deletion mutants for analysis ofWnt1-based tumor evolution.

ZFN-Based Gene Editing in Producing DKK3b Reporter Mouse and anyPromoter Function Studies

The zinc-finger nuclease (ZFN)-based gene editing approach is used toproduce a Dkk3b reporter mouse by redirecting the DKK3b promoter todrive expression of a fluorescent reporter (FIG. 4).

During validation of the ZFN pairs, gene-edited, Dkk3b-promoter drivenCFP C8 (C8Dkk3b^(CFP+/−)) cells are generated by default. The C8astrocyte is an immortal cell line with suppressed Dkk3 gene expressionand can be used to explore the cellular event(s) impacted by DKK3bwithout the confounding impact of aneuploidy/polyploidy and the geneticinstability inherent in tumor cell lines. Moreover, this cell line is aninvaluable resource for promoter function studies. Systematic functionalanalysis of the Dkk3b promoter is done to identify individual Dkk3bpromoter elements using CFP expression as the endpoint and in silicoanalysis (TranFac, Genomatrix algorithms) of promoter element(s). One ormore promoter modulators that increase transcription from this locus areidentified using this promoter surveillance approach.

The gene edited Dkk3b locus is markedly “silenced” by hypermethylationin the immortalized C8 cells. Methylase inhibitors and deacetylationactivators were used to increase expression from this promoter. As shownin FIG. 12, the gene edited C8^(Dkk3bCFP+/−) cells expressed weak CFPsignals, while inhibition of methylation markedly increased reporterexpression. These data validate the ZFN targeting strategy, confirm CFPinsertion and show that methylase inhibitors can derepress the“silenced” DKK3 promoter.

DKK3b Fusion Protein and Effects on Beta-Catenin Dependent GeneExpression, Cell Proliferation and Metastasis

Small membrane transduction domains (MTD) are fused to the N-terminus ofDKK3b to produce a bioavailable fusion protein using published methods.(Nagahara, et al. 1998 Nature medicine 4(12):1449-52.). A TAT-DKK3bfusion construct was assembled and purified the fusion protein fromurea-denatured bacterial lysates using Ni-NTA resins. FIG. 13A shows theepitope domains of the TAT-DKK3b fusion protein. As shown in FIG. 13B, a5 min treatment of Wnt-stimulated NMuMG and HEK293T cells with TAT-DKK3b(≥40 fg/cell) silenced β-catenin-dependent TCF-, E2F- and RelA-drivengene expression and restored expression of β-catenin-suppressed markersof cellular differentiation, E-cadherin and Elf3 (FIG. 13B). The data inFIG. 14 shows that TAT-DKK3b treatment had no effect on untransformedNMuMG cells, but arrested cell proliferation of Wnt-stimulated cells.TAT-DKK3b also blocked migration of the highly invasive human MDA-MB-231breast cancer cells. These data show that the bacterially expressed,membrane-permeant DKK3b retains all of the anti-cancer properties ofnative DKK3b.

The TAT-HA-DKK3b fusion protein used in initial studies included epitopetags and ancillary sequence that comprised almost 25% of the fusionprotein and are undesirable in a therapeutic product. To reduceantigenicity and eliminate unnecessary sequence, the TAT-HA cassette isreplaced with an 11 residue long synthetic MTD reported to increasemembrane permeability by >30 fold reduce/eliminate antigenicity and toextend the biological half-life of the encoded fusion protein. Theoptimized MTD-DKK3b will retain the polyHistidine tag for purificationof the MTD-DKK3b under denaturing conditions.

Optimize Conditions for Production of MTD-DKK3b in Bacteria.

Small cultures of the individual pMTD-DKK3b constructs are prepared inIPTG-inducible, T7 polymerase expressing E. coli. IPTG-induction/growthconditions are chosen so that >90% of the MTDDKK3b is localized toinclusion bodies. Bacterial cells are urea extracted, MTD-DKK3b purifiedby Ni++ affinity isolation and then formulated without urea. MTD-DKK3bexpression levels are evaluated by immunoblot analysis usinganti-polyHis or anti-DKK3b antibodies. Purified candidate MTD-DKK3bs aretested for purity by SDS-PAGE and SEC-HPLC, and for bioactivity usingreporter cell lines that secrete b-catenin dependent reporters into thegrowth medium (see below). The optimal clone are used to produce aMaster Cell Bank for future clinical development.

Develop Purification Scheme for Unfolded MTD-DKK3b Using GMP Standards.

Bacterial cell lysis and isolation of the MTD-DKK3b from inclusionbodies are systematically optimized. The concentration of denaturants,urea and/or chaotropic salts, lytic conditions and cleanup steps aresystemically examined Urea concentrations are optimized for maximumrecovery of the fusion protein while minimizing the urea content of theextraction buffers for ease of purification scalability.

Urea extracted proteins are affinity purified on Ni⁺⁺ resins and elutedwith imidazole in buffered urea. Urea is then rapidly removed tominimize refolding of the MTDDKK3b fusion protein. Optimization ofpurification at this stage primarily involves investigation of washingsteps while the protein is bound to the Ni⁺⁺ resin with variable washbuffer pH and imidazole concentrations. A key parameter at this stage isthe reduction of contamination. Bioactivity of the purified MTD-DKK3b isevaluated b-catenin dependent reporter cell lines. Refolding andaggregation are monitored by fluorescent dye binding.

Further information on assembly of TAT/PTD DKK3b are provided in FIGS.15-22.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

The invention claimed is:
 1. A method of inhibiting β-catenin signalingto treat a patient suffering from a cancer or a tumor comprisingadministering to the patient a composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a fusionprotein that is capable of preventing β-catenin from reaching itsnuclear TCF target, wherein the fusion protein comprises a membranetransduction domain (MTD) peptide fused to the N-terminus of the humanintracellular Dickkopf (Dkk3b) protein encoded by exons 3-8 of the humanDkk3 gene locus.
 2. The method of claim 1, wherein the MTD comprises theamino acids 5-15 of SEQ ID NO:
 7. 3. The method of claim 1, wherein theMTD comprises TAT.
 4. The method of claim 1, wherein the cancer isselected from the group consisting of, lymphoma, blastoma, sarcoma,liposarcoma, neuroendocrine tumor, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cellcancer, epithelial squamous cell cancer, lung cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophageal cancer, a tumor of the biliary tract, and head and neckcancer.
 5. A method for inhibiting tumor progression in a patient inneed thereof, comprising administering to the patient a compositioncomprising a fusion protein comprising a membrane transduction domain(MTD) peptide fused to the N-terminus of the human intracellularDickkopf (Dkk3b) protein encoded by exons 3-8 of the human Dkk3 genelocus and a pharmaceutically acceptable carrier, wherein the tumor hasincreased β-catenin and/or Wnt signaling, and wherein the fusion proteininhibits β-catenin and/or Wnt signaling thereby inhibiting tumorprogression in the patient.
 6. The method of claim 5 wherein the MTDcomprises the amino acids 5-15 of SEQ ID NO:
 7. 7. The method of claim 5wherein the MTD comprises TAT.
 8. The method of claim 1, wherein thepatient is suffering from a tumor.
 9. The method of claim 8, wherein thetumor is selected from the group consisting of prostate cancer breastcancer, lung cancer, gastrointestinal cancer, ovarian cancer, kidney orrenal cancer, and pancreatic cancer.
 10. The method of claim 4, whereinthe cancer is selected from the group consisting of prostate cancer,breast cancer, lung cancer, gastrointestinal cancer, ovarian cancer,kidney or renal cancer, and pancreatic cancer.